This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our INBRE supported research is focused on generating a better understanding of zinc-containing hydrolases involved in bacterial proliferation. Systems currently under study include several metallo-beta-lactamases, which confer resistance to beta-lactam antibiotics, such as penicillin, and the quorum-sensing AHL lactonase from Bacillus thuringiensis, which functions in intercellular communication within bacterial colonies. Each catalytic site utilizes one or two zinc ions, making them difficult to study spectroscopically. Aside from crystallography, X-ray absorption spectroscopy (XAS) is the only method for direct interrogation of local structure in a Zn-containing enzyme. We are using XAS to study native, Zn-containing enzymes in their resting states and, where available, product-bound forms. To open these systems to a wider array of spectroscopic studies, it is necessary to incorporate a paramagnetic metal ion, such as Co(II). We utilize XAS as an equally critical component of the study of Co-substituted enzymes, providing an independent comparison with the local structure of the native Zn-enzymes.